The art is generally cognizant of the basic technique of fusing mouse myeloma cells to spleen cells from immunized mice to obtain a culturable, continuous cell line capable of producing homogeneous or "monoclonal" antibodies. See, for example, Kohler and Milstein, Nature 256, 495-497 (1975). Attempts to apply this general knowledge are often frustrated by particular problems and difficulties encountered with regard to particular antigens. Furthermore, the complexity of such antigens, the almost inevitable inclusion in inocula of other, accidentally associated antigenic material, and the existence of various incidental immunities in the mice whose spleen cells are used prevent any assurance that the monoclonal antibody eventually produced will in fact be to the target antigen.
In biological systems, complex antigens commonly change as they move from one context to another within an organism. A monoclonal antibody to the antigen may recognize it only in one form. Likewise, when the antigen for which a monoclonal antibody is desired appears in corresponding forms in various species, it is not uncommon that the antigen varies slightly from species to species. Consequently, there is no assurance that any particular cross species specificity will be obtained or that even within a given species an antigen will be recognized in any particular biological context.
In the production of monoclonal antibodies and the hybridomas making them, a convenient experimental animal, such as a mouse, is exposed to the antigen against which an antibody is desired. Typically, some of the antigen is injected into the animal, and its immune system is allowed to respond to it. This process may be repeated until the animal's immune system is presumed to be producing antibodies to the antigen, as well as such other antibodies as the animal may be producing without regard to the injections of the antigen. The animal is then killed, and antibody-producing cells from it are isolated. Typically spleen cells from the animal are employed.
A large number of such spleen cells are then fused with myeloma cells of the same species to obtain hybrid cells that will reproduce without the self-limiting growth characteristics of most non-tumor cells. The fused cells are then cultured as cell lines of genetically identical, antibody-producing cells. However, there is no assurance that the antibody produced by any particular cell line is an antibody to the original antigen or that the antibody will be specific to the antigen. In order to select from among the many hybridoma cell lines thus created for a particular cell line that produces a desired antibody, it is necessary to screen the cell lines. This is done by testing the antibody produced by each cell line against the original antigen or a purified form thereof. The cell lines that are found by this means to produce the desired antibody are then preserved, and the remainder are discarded.
It should be emphasized again that the unpredictable nature of hybrid cell preparation generally does not allow one to extrapolate from one antigen or cell system to another in order to predict precise outcomes of the application of conventional hybridization techniques. This unpredictability is further increased as the antigen is more complex and as an antibody is sought capable of recognizing the antigen in more than one species or in more than one form or context within a biological system.
Thrombospondin is the major glycoprotein released from alpha granules of thrombin-stimulated blood platelets. In addition, thrombospondin is synthesized by growing cells. See Mosher, Doyle, and Jaffe, "Synthesis and Secretion of Thrombospondin by Cultured Human Endothelial Cells," J. Cell Biol. 93, 343-348 (1982); Raugi et al., "Thrombospondin: Synthesis and Secretion by Cells in Culture," J. Cell Biol. 95, 351-354 (1982).
Blood platelet alpha granules are membrane-enclosed sacs contained within the body of the platelet. Each platelet has a number of alpha granules. Alpha granules contain a variety of proteins including thrombospondin, platelet factor 4, and beta thromboglobulin. In addition, other proteins are contained within alpha granules that are related to or identical with certain plasma proteins that take part in blood clotting, such as fibrinogen and accelerin (blood coagulation factor V). When platelets are stimulated by thrombin, for example as part of the body'esponse to trauma to a blood vessel, the alpha granules discharge their contents into the blood, the proteins contained therein participating further in various ways in the aggregation of platelets and related processes.
In various contexts, it is useful to be able to determine routinely the presence and the quantity of thrombospondin in a sample of material. For example, fetal calf serum is a commercially available material extensively used in cell culturing and other experimental and commercial operations. As is the case with any material used in sensitive procedures, it is important to be able accurately to characterize fetal calf serum. For example, it is important to determine the amount of particular materials that are present in fetal calf serum in varying quantities, depending upon the source of the serum and the conditions of preparation. The proteins released from platelet alpha granules are an example of materials that are present in varying quantities in fetal calf serum. Currently tests for platelet factor 4 and beta thromboglobulin are utilized not just to determine the presence of those materials but as an indication of recent platelet activation in human patients, since both of those materials form part of the contents of alpha granules which are released during platelet activation. However, tests for these materials in blood plasma have not proved reliably sensitive and specific for alpha granule release. It is speculated that these two materials are rapidly cleared from the circulation once released from platelets, making it impossible to precisely relate their concentrations to the likely concentrations of other materials released with them from alpha granules. The ability to test quantitatively for the presence of thrombospondin would provide an additional and probably superior means to test for alpha granule release.
A reliable, specific, and quantitative test for thrombospondin would be an important research tool with various applications. For example, there are indications that elevated levels of thrombospondin in blood plasma may be characteristic of disseminated intravascular coagulation associated with septicemia, thrombotic thrombocytopemia purpura, cancers, and neoplasms. Quantitative assays for thrombospondin would have application in studies of these conditions and in the examination of lysed platelets and of joint fluids from people with various types of arthritic conditions and other joint diseases or abnormalities. A monoclonal antibody specific to thrombospondin would further allow the specific measurement of thrombospondin in a variety of biological fluids and cell and tissue extracts, for example by enzyme-linked immuno-sorbent assays, (hereinafter "ELISAs") and other immunoassays. In other contexts, such an antibody would allow locating thrombospondin in a tissue section by immunofluorescence and other immunohistological techniques. In vivo the presence of thrombospondin could be detected by nuclear scanning for a radiolabeled version of such an antibody.
It is possible to produce anti-thrombospondin antibodies by conventional innoculation of rabbits or other animals followed by the processing of serum later extracted from the innoculated animal. The attempted production of a monoclonal antibody to thrombospondin has not been reported.